The broad, long-term objective of my laboratory is a detailed molecular understanding of how cell fates are regulated by division sequence and cell-cell interactions in animal development. Wnt signaling is required for the fate difference between anterior- posterior (a-p) sisters, MS and E, in early C. elegans embryos. The nuclear factor POP-1 functions downstream in Wnt signaling to specify the fate of the anterior cell, MS. POP-1 protein is at a' higher level in MS than in E, and this asymmetric POP-I pattern requires Wnt signaling. In addition to MS)E, higher levels of POP- 1 are observed in all anterior sisters of a-p divisions in early embryos and this asymmetry appears to be required for a-p fate differences in the lineages examined. Many Wnt signaling components are not required for this global POP-1 asymmetry. The hypothesis to be tested are: (1) POP-1 levels are a readout for a-p polarity. (2) Global POP-1 asymmetry is regulated by a mechanism distinct from Wnt signaling. (3) In selected a-p sisters, additional signaling is integrated with the mechanism that regulates global POP-1 asymmetry to fine-tune the polarity. The specific aims of this proposal are: (1) characterization and cloning of a novel gene pie-3. We have recently isolated a mutant in the gene pie-3 defective in POP-1 asymmetry throughout the embryo. Phenotypic and molecular characterization of pie-3 should reveal insight into the regulation of global POP-1 asymmetry. (2) generation and characterization of null and temperature-sensitive mutations in the Pop-1 gene. If POP-1 levels are a readout of a-p polarity, one would expect POP-1 to function at each division to regulate a-p sister fates. Generation of pop-1 null and ts mutations will allow us to examine the function of pop-1 at multiple developmental stages. (3) screen for conditional mutants in Wnt signaling. Many of the Wnt signaling genes regulating the MS/E fate decision might also function at other developmental stages and are not likely to be identified in traditional screens. We developed the en masse slide screen to isolate ts mutants defective in Wnt signaling. (4) identification of genes regulating POP-1 asymmetry. We will use a POP-1-GFP reporter fusion that recapitulates the expression pattern of POP-1 to identify genes important for POP-1 asymmetry by screening directly for mutants defective in POP-1 distribution. We will use the en masse slide screen to isolate mutants defective in POP-1 asymmetry either globally or within a particular lineage. This should identify genes involved in the establishment, and regeneration of POP-1 asymmetry. These experiments will advance our understanding of developmental pathways that, like Wnt signaling, regulate key developmental processes in the vertebrate embryo.